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Abstract:

The present invention relates to Polycomb Repressive Complex 2 (PRC2)
peptide inhibitors and their use for the treatment of cancer and other
conditions associated with aberrant PRC2 methyltransferase activity.

Claims:

2. The isolated peptide of claim 1, wherein X at position 1 is a lysine
or arginine residue, X at position 7 is an alanine or serine residue, and
X at position 9 and position 10 is a serine, alanine, or threonine
residue.

3. The isolated peptide of claim 1, wherein said peptide is less than 100
amino acid residues in total length.

4. The isolated peptide of claim 1, wherein said peptide is between 11-50
amino acid residues in length.

11. The fusion peptide of claim 7, wherein the targeting moiety is a
nuclear localization moiety.

12. A pharmaceutical composition comprising: the isolated peptide of
claim 1 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12 further comprising: a
delivery vehicle.

14. A method of treating a subject having cancer comprising: selecting a
subject having cancer and administering an isolated peptide comprising an
amino acid sequence of XAARMSXPXXG (SEQ ID NO: 4), wherein X is any amino
acid residue, to the subject under conditions effective to treat the
cancer.

15. The method of claim 14, wherein said cancer is mediated by aberrant
PRC2 activity.

16. The method of claim 14, wherein X at position 1 of SEQ ID NO: 4 is a
lysine or arginine residue, X at position 7 is an alanine or serine
residue, and X at position 9 and position 10 is a serine, alanine, or
threonine residue.

17. The method of claim 14, wherein said peptide is less than 100 amino
acid residues in total length.

[0006] A first aspect of the present invention relates to an isolated
peptide comprising an amino acid sequence of XAARMSXPXXG (SEQ ID NO:1),
wherein X is any amino acid residue.

[0007] Another aspect of the present invention relates to a method of
treating a subject having cancer that involves selecting a subject having
cancer and administering an isolated peptide comprising an amino acid
sequence of XAARMSXPXXG (SEQ ID NO:1), wherein X is any amino acid
residue, to the subject under conditions effective to treat the cancer.

[0008] Recent exome sequencing studies of Pediatric Diffuse Pontine
Gliomas (DIPG) and Glioblastoma Multiforme (GBM) identified the missense
mutation K27M in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B).
The heterozygous nature of these mutations suggests that they promote
gliomagenesis through a gain-of-function mechanism that is not
understood. As shown herein, expression of H3.1 or H3.3 transgenes with
the K27M mutation leads to a striking reduction of H3K27me3 on the
non-mutated H3 both within the same nucleosome and on nearby nucleosomes.
This reduction is specific for the methionine substitution at position 27
as transgenes encoding K27R or K27Q do not cause lower K27me3 levels. The
Polycomb Repressive Complex 2 (PRC2) shows decreased methyltransferase
activity on nucleosome templates containing H3K27M. These and other data
led to the discovery of isolated peptides derived from H3K27M that
function as potent PRC2 inhibitors. To date, few, if any, inhibitors for
PRC2 exist, despite active investigation by academic, biotech, and
pharmaceutical entities into the link between PRC2 activity, H3K27
methylation and the variety of human cancers cited above.

[0010] FIGS. 2A-2D demonstrate that H3K27M-containing chromatin is a poor
substrate for PRC2. FIG. 2A is a silver stained gel of PRC2 used for in
vitro histone methyltransferase (HMT) reactions. FIG. 2B is a coomassie
stained gel of purified mono- or oligo-nucleosomes used for in vitro PRC2
histone methyltransferase reactions. FIG. 2C shows that H3K27M-containing
chromatin templates are poor substrates for PRC2-dependent methylation.
Shown is a fluorograph of in vitro HMT assay with purified PRC2 and
chromatin templates show in FIGS. 2A and 2B. The decrease in
PRC2-dependent methylation is specific for H3K27M chromatin as shown in
the fluorograph of FIG. 2D. Fluorograph from in vitro HMT assay with
purified mononucleosomes containing H3K27M, K27R, K27A or K27Q and PRC2.

[0011] FIGS. 3A-3F show inhibition of PRC2 activity by H3K27M peptide in
trans. FIGS. 3A and 3B show titration of various H3 peptides (18-37) into
in vitro HMT assay with PRC2 and purified wildtype (FIG. 3A) or
H3K27M-containing mononucleosomes (FIG. 3B). FIG. 3C shows the
quantitation of HMT assays on purified mononucleosome substrates.
Unmodified (18-37), K27ac or K27M missense mutations in H3F3A and
HIST3H1B were verified by DNA sequencing. As shown in FIG. 3D, increased
H3K27ac/H3K27me3 peptide ratio does not affect allosteric activation of
PRC2 by H3K27me3 peptide. An increased ratio of H3K27M/H3K27me3 peptide
ratio decreases H3K27me3-dependent allosteric activation or PRC2 as shown
in FIG. 3E. FIG. 3F shows inhibition of PRC2 activity by titration of
H3K27M, but not H3K27ac peptides.

[0012] FIGS. 4A-4B demonstrate that H3K27M peptides exhibit mixed
non-competitive inhibition of PRC2 methyltransferase activity. In the
graph of FIG. 4A the initial reaction rates from assays with PRC2 were
determined at various nucleosome concentrations. The assays were
performed without, and with the H3K27M peptide at two concentrations.
FIG. 4B is a double-reciprocal Lineweaver-Burke plot corresponding to the
data in FIG. 4A.

[0016] Variants of the isolated peptides derived from SEQ ID NOs: 2 or 3
are also encompassed by the present invention. Suitable variant peptides
include those peptides having one or more amino acid substitutions that
retain the ability to inhibit PRC2 methyltransferase activity. More
specifically, the present invention encompasses any isolated variant
peptides that inhibit PRC2 mediate H3K27 tri-methylation (H3K27me3).
Inhibitory functionality of a variant peptide can readily be assessed
using various assays, including, for example and without limitation, an
in vitro H3K27 methyltransferase assay as described in the Examples
herein.

[0017] A variant peptide of the present invention may contain one or more
amino acid residue additions, deletions, or substitutions. An isolated
variant peptide of the present invention retains at least about 30-50%
sequence identity to the amino acid sequence of H3.1 or H3.3 from which
it is derived from. Preferably, variant peptides retain at least 60-70%
or 70-80% sequence identity to the amino acid sequence of H3.1 or H3.3.
More preferably, variant peptides retain at least 80-90% sequence
identity to the amino acid sequence H3.1 or H3.3. Most preferably,
variant peptides of the present invention retain 90-95% or 95-99%
sequence identity to the amino acid sequence of the H3.1 or H3.3 which it
is derived from.

[0018] When a variant peptide of the present invention comprises amino
acid substitutions, such substitutions preferably comprise conservative
natural or non-natural amino acid substitutions. Conservative amino acid
substitutions may include synonymous amino acid residues within a group
which have sufficiently similar physicochemical properties, so that a
substitution between members of the group will preserve the biological
activity of the molecule (see e.g. Grantham, R., "Amino Acid Difference
Formula to Help Explain Protein Evolution," Science 185: 862-864 (1974),
which is hereby incorporated by reference in its entirety). It is evident
that amino acids may also be inserted and/or deleted in the above-defined
sequences without altering their function, particularly if the insertions
and/or deletions only involve a few amino acids, e.g. less than 5 to 10,
and preferably less than 2 to 5, and do not remove or displace amino
acids which are critical to functional activity (i.e., the core AARMS
sequence). Synonymous amino acid residues are identified in Table 1
below. Other conservative interchanges include those within the aliphatic
group aspartate and glutamate; within the amide group asparagine and
glutamine; within the hydroxyl group serine and threonine; within the
chromatic group phenylalanine, tyrosine and tryptophan; within the basic
group lysine, arginine and histidine; and within the sulfur-containing
group methionine and cysteine. Sometimes substitution within the group
methionine and leucine can also be considered conservative. Preferred
conservative substitution groups are aspartate-glutamate;
asparagine-glutamine; valine-leucine-isoleucine; alanine-valine;
phenylalanine-tyrosine; and lysine-arginine.

[0020] In another embodiment of the present invention, the isolated
peptide comprises an amino acid sequence of TXAARMSXPXXGGVK (SEQ ID NO:
41), where X at positions 2, 8, 10, and 11 comprises any amino acid
residue. Alternatively, X at position 2 of SEQ ID NO: 41 comprises a K or
R residue, X at position 8 of SEQ ID NO: 41 comprises an A or S residue,
and X at positions 10 and 11 of SEQ ID NO: 41 comprises a S, A, or T
residue. As can be appreciated by one of skill in the art, isolated
peptides in accordance with this embodiment of the present invention
comprise any one of the amino acid sequences shown in Table 2 above
flanked by a T residue at the amino-terminus and GVK residues at the
carboxy-terminus. Exemplary isolated peptides of the present invention
comprise, without limitation, the amino acid sequence of TKAARMSAPATGGVK
(SEQ ID NO: 42) or TKAARMSAPATGGVK (SEQ ID NO: 43). The present invention
further relates to isolated nucleic acid molecules encoding the peptides
of the present invention as described in more detail infra.

[0021] In another embodiment of the present invention, the isolated
peptide comprises an amino acid sequence of KQLATXAARMSXPXXGGVKK (SEQ ID
NO: 44), X at positions 6, 12, 14, and 15 comprises any amino acid
residue. Alternatively, X at position 6 of SEQ ID NO: 44 comprises a K or
R residue, X at position 12 of SEQ ID NO: 44 comprises an A or S residue,
and X at positions 14 and 15 of SEQ ID NO: 44 comprises a S, A, or T
residue. As can be appreciated by one of skill in the art, isolated
peptides in accordance with this embodiment of the present invention
comprise any one of the amino acid sequences shown in Table 2 above,
flanked by KQLAT residues at the amino-terminus and GVKK residues at the
carboxy-terminus. Exemplary isolated peptides of the present invention
comprise, without limitation, the amino acid sequence of
KQLATKAARMSAPATGGVKK (SEQ ID NO: 45) or KQLATKAARMSAPSTGGVKK (SEQ ID NO:
46). The present invention further relates to isolated nucleic acid
molecules encoding the peptides of the present invention as described in
more detail infra.

[0022] The isolated peptides of the present invention may be composed
exclusively of L-amino acids, D-amino acids, or any combination thereof.
Peptides comprising D-amino acids are advantageous because they are less
susceptible to degradation, enter cells as easily as an L-amino acid
peptide, are easy to synthesize, and in some cases are less antigenic. A
peptide of the present invention comprising D-amino acids preferably
comprises a D-retro-inverso-peptide sequence corresponding to its L-amino
peptide sequence counterpart. A "retro-inverso sequence" is an isomer of
a linear peptide sequence in which the direction of the sequence is
reversed and the chirality of each amino acid residue is inverted (see
e.g., Jameson et al., "A Rationally Designed CD4 Analogue Inhibits
Experimental Allergic Encephalomeylitis," Nature 368:744-746 (1994) and
Brady et al., "Drug Design. Reflections on a Peptide," Nature 368:692-693
(1994), which are hereby incorporated by reference in their entirety).
The advantage of combining D-enantiomers and reverse synthesis is that
the positions of carbonyl and amino groups in each amide bond are
exchanged, while the position of the side-chain groups at each alpha
carbon is preserved. Unless specifically stated otherwise, it is presumed
that any given L-amino acid sequence or peptide according to the present
invention may be converted into an D-retro-inverso sequence or peptide by
synthesizing a reverse of the sequence or peptide for the corresponding
native L-amino acid sequence or peptide.

[0023] The isolated peptides of the present invention are preferably
acetylated at the N-terminus and amidated at the carboxy terminus to
increase cell permeability and enhance peptide stability. Methods of
N-terminal acetylation and C-terminal amidation of synthetic peptides are
well known in the art.

[0024] Another aspect of the present invention relates to fusion peptides
comprising the isolated peptide described herein and a targeting moiety
that is coupled to the isolated peptide. Suitable targeting moieties
include cell specific targeting moieties, cell-penetrating moieties, and
intracellular localization or trafficking moieties. One or more targeting
moieties can be coupled to the amino and/or carboxy termini of the
isolated peptide of the invention.

[0026] In another embodiment, the targeting moiety comprises a sequence
that directs cell uptake of the peptide. For example, the targeting
moiety can be derived from a known membrane-translocating sequence, such
as the sequence for human immunodeficiency virus (HIV)-1 trans-activator
of transcription (TAT) protein (see e.g., U.S. Pat. No. 5,804,604 to
Frankel et al, and U.S. Pat. No. 5,674,980 to Frankel et al., which are
hereby incorporated by reference in their entirety). An isolated peptide
of the present invention may be coupled to the 86 amino acid residue TAT
protein or a fragment thereof. Preferably, a functionally effective
fragment or portion of a TAT protein that has fewer than 86 amino acids,
exhibits uptake into cells, and optionally uptake into the cell nucleus
is utilized. In one embodiment, the TAT peptide comprises amino acid
residues 48-57, e.g. NH2-GRKKRRQRRR-COOH (SEQ ID NO: 47), a generic
TAT sequence NH2-Xn-RKKRRQRRR-Xn-COOH (SEQ ID NO: 48), or
a D-retro-inverso peptide having the sequence
NH2-Xn-RRRQRRKKR-Xn-COOH (SEQ ID NO: 49). A TAT peptide
that includes the region that mediates entry and uptake into cells can be
further defined using known techniques (see e.g., Frankel et al,
"Activity of Synthetic Peptides from the Tat Protein of Human
Immunodeficiency Virus Type-1," Proc. Natl. Acad. Sci. USA 86: 7397-7401
(1989), which is hereby incorporated by reference in its entirety).

[0027] The TAT sequence may be coupled to the N-terminal or the C-terminal
end of the isolated peptide of the present invention. A hinge of two
proline residues may be added between the TAT and the isolated peptide of
the present invention to create a fusion peptide. Alternatively, the TAT
sequence can be linked to the isolated peptide of the present invention
using other suitable linker sequences, such as, glycine-rich (e.g.
G3-5) or serine-rich (e.g., GSG, GSGS (SEQ ID NO: 50), GSGSG (SEQ ID
NO: 51), GSNG) linker sequences, or flexible immunoglobulin linkers
as disclosed in U.S. Pat. No. 5,516,637 to Huang et al, which is hereby
incorporated by reference in its entirety.

[0028] The TAT targeting moiety can be a single (i.e., continuous) amino
acid sequence present in the TAT sequence. Alternatively it can be two or
more amino acid sequences, which are present in TAT protein, but are not
contiguous in the naturally-occurring TAT protein. Modifications to TAT
protein and fragments thereof designed to modulate intracellular
localization and/or enhance membrane solubility are further described in
U.S. Pat. No. 5,804,604 to Frankel et al, and U.S. Pat. No. 5,674,980 to
Frankel et al., which are hereby incorporated by reference in their
entirety. TAT protein can be obtained from naturally-occurring sources or
can be produced using genetic engineering techniques or chemical
synthesis.

[0029] Another suitable targeting moiety useful for promoting the cellular
uptake of an isolated peptide of the present invention comprises a cell
penetrating peptide (CPP). CPPs translocate across the plasma membrane of
eukaryotic cells by a seemingly energy-independent pathway and have been
used successfully for intracellular delivery of macromolecules, including
antibodies, peptides, proteins, and nucleic acids, with molecular weights
several times greater than their own. Several commonly used CPPs,
including polyarginines, transportant, protamine, maurocalcine, and M918,
are suitable targeting moieties for use in the present invention and are
well known in the art (see Stewart et al., "Cell-Penetrating Peptides as
Delivery Vehicles for Biology and Medicine," Organic Biomolecular Chem
6:2242-2255 (2008), which is hereby incorporated by reference in its
entirety). Additionally, methods of making CPP are described in U.S.
Patent Application Publication No. 20080234183 to Hallbrink et al., which
is hereby incorporated by reference in its entirety.

[0030] Another suitable targeting moiety useful for enhancing the cellular
uptake of an isolated peptide of the present invention is an "importation
competent" signal peptide as disclosed by U.S. Pat. No. 6,043,339 to Lin
et al., which is hereby incorporated by reference in its entirety. An
importation competent signal peptide is generally about 10 to about 50
amino acid residues in length, typically hydrophobic residues, which
render the peptide capable of penetrating through the cell membrane from
outside the cell to the interior of the cell. An exemplary importation
competent signal peptide includes the signal peptide from Kaposi
fibroblast growth factor (see U.S. Pat. No. 6,043,339 to Lin et al.,
which is hereby incorporated by reference in its entirety). Other
suitable peptide sequences can be selected from the SIGPEP database (see
von Heijne G., "SIGPEP: A Sequence Database for Secretory Signal
Peptides," Protein Seq. Data Anal. 1(1):41-42 (1987), which is hereby
incorporated by reference in its entirety).

[0031] Another suitable targeting sequence or moiety is a transport
peptide that directs intracellular compartmentalization of the isolated
peptide once it is internalized by a target cell or tissue. For example,
to achieve nuclear localization, the isolated peptide of the present
invention is coupled to a nuclear localization transport signal. Suitable
nuclear transport peptide sequences are known in the art, including the
nuclear transport peptide PPKKKRKV (SEQ ID NO: 52). Other nuclear
localization transport signals include, for example, the nuclear
localization sequence of acidic fibroblast growth factor, the nuclear
localization sequence of the transcription factor NF-KB p50 (U.S. Pat.
No. 6,043,339 to Lin et al., which is hereby incorporated by reference in
its entirety), and the intracellular trafficking sequence derived from
the Herpesvirus structural VP22 protein (WO 97/05265 to O'Hare and
Elliott and O'Hare, "Intracellular Trafficking and Protein Delivery by a
Herpesvirus Structural Protein," Cell 88: 223-233 (1997), which are
hereby incorporated by reference in their entirety.

[0032] The targeting moiety can be coupled to the isolated peptide of the
present invention by chemical coupling in any suitable manner known in
the art. In one embodiment, the chemical cross-linking method is a
non-specific method, i.e. the point of coupling is not directed to any
particular site on the transport or cargo peptide or polypeptide.
Alternatively, the targeting moiety can be directly coupled to the
isolated peptide of the present invention via a functional group (e.g.,
cysteine residue or primary amine), found only once or a few times in one
or both of the targeting moiety and cargo peptide to be cross-linked.

[0033] Coupling of the two constituents can be accomplished via a coupling
or conjugating agent. There are several intermolecular cross-linking
reagents which can be utilized, e.g., J-succinimidyl 3-(2-pyridyldithio)
propionate (SPDP) or N,N'-(1,3-phenylene) bismaleimide (both of which are
highly specific for sulfhydryl groups and form irreversible linkages);
N,N'-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11
carbon methylene bridges (which are relatively specific for sulfhydryl
groups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversible
linkages with amino and tyrosine groups). Other cross-linking reagents
useful for this purpose include, without limitation,
p,p'-difluoro-m,m'-dinitrodiphenylsulfone (which forms irreversible
cross-linkages with amino and phenolic groups); dimethyl adipimidate
(which is specific for amino groups); phenol-1,4-disulfonylchloride
(which reacts principally with amino groups); hexamethylenediisocyanate
or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts
principally with amino groups); glutaraldehyde (which reacts with several
different side chains) and disdiazobenzidine (which reacts primarily with
tyrosine and histidine). Heterobifunctional cross-linking agents having
two different functional groups, e.g., amine- and thiol-reactive groups,
that will cross-link two proteins having free amines and thiols,
respectively, are also suitable cross-linking agents. Examples of
heterobifunctional cross-linking agents include succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, and succinimide
4-(p-maleimidophenyl) butyrate. The succinimidyl group of these
cross-linkers reacts with a primary amine, and the thiol-reactive
maleimide forms a covalent bond with the thiol of a cysteine residue.

[0034] In one embodiment of the present invention, a cross-linking reagent
that forms a cleavable covalent bond (e.g., disulfide bond) under
cellular conditions is preferentially utilized. Exemplary cross-linking
agents that form cleavable bonds include, without limitation, Traut's
reagent, dithiobis (succinimidylpropionate), and N-succinimidyl
3-(2-pyridyldithio) propionate. The use of a cleavable cross-linking
reagent permits the cargo moiety (i.e., the PCR2 inhibitory peptide) to
separate from the transport polypeptide after delivery into the target
cell.

[0035] The isolated peptides of the present invention may be prepared
using standard methods of synthesis known in the art, including solid
phase peptide synthesis (Fmoc or Boc strategies) or solution phase
peptide synthesis. Alternatively, peptides of the present invention may
be prepared using recombinant expression systems as described below.

[0036] Generally, the use of recombinant expression systems involves
inserting the nucleic acid molecule encoding the amino acid sequence of
the desired peptide into an expression system to which the molecule is
heterologous (i.e., not normally present). Isolated nucleic acid
molecules encoding the peptides of the present invention can be derived
from the nucleotide sequences encoding H3.1 (SEQ ID NO: 53) and H3.3 (SEQ
ID NO: 54) shown below.

[0037] One or more desired nucleic acid molecules encoding a peptide of
the invention may be inserted into the vector. When multiple nucleic acid
molecules are inserted, the multiple nucleic acid molecules may encode
the same or different peptides. The heterologous nucleic acid molecule is
inserted into the expression system or vector in proper sense
(5'→3') orientation relative to the promoter and any other 5'
regulatory molecules, and correct reading frame.

[0038] The preparation of the nucleic acid constructs can be carried out
using standard cloning procedures well known in the art as described by
Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold
Springs Harbor 1989). U.S. Pat. No. 4,237,224 to Cohen and Boyer, which
is hereby incorporated by reference in its entirety, describes the
production of expression systems in the form of recombinant plasmids
using restriction enzyme cleavage and ligation with DNA ligase. These
recombinant plasmids are then introduced by means of transformation and
replicated in a suitable host cell.

[0039] A variety of genetic signals and processing events that control
many levels of gene expression (e.g., DNA transcription and messenger RNA
("mRNA") translation) can be incorporated into the nucleic acid construct
to maximize peptide production. For the purposes of expressing a cloned
nucleic acid sequence encoding a desired peptide, it is advantageous to
use strong promoters to obtain a high level of transcription. Depending
upon the host system utilized, any one of a number of suitable promoters
may be used. For instance, when cloning in E. coli, its bacteriophages,
or plasmids, promoters such as the T7 phage promoter, lac promoter, trp
promoter, recA promoter, ribosomal RNA promoter, the PR and PL
promoters of coliphage lambda and others, including but not limited, to
lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels
of transcription of adjacent DNA segments. Additionally, a hybrid
trp-lacUV5 (tac) promoter or other E. coli promoters produced by
recombinant DNA or other synthetic DNA techniques may be used to provide
for transcription of the inserted gene. Common promoters suitable for
directing expression in mammalian cells include, without limitation,
SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early,
immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.

[0040] There are other specific initiation signals required for efficient
gene transcription and translation in prokaryotic cells that can be
included in the nucleic acid construct to maximize peptide production.
Depending on the vector system and host utilized, any number of suitable
transcription and/or translation elements, including constitutive,
inducible, and repressible promoters, as well as minimal 5' promoter
elements, enhancers or leader sequences may be used. For a review on
maximizing gene expression see Roberts and Lauer, "Maximizing Gene
Expression On a Plasmid Using Recombination In Vitro," Methods in
Enzymology 68:473-82 (1979), which is hereby incorporated by reference in
its entirety.

[0041] A nucleic acid molecule encoding an isolated peptide of the present
invention, a promoter molecule of choice, including, without limitation,
enhancers, and leader sequences; a suitable 3' regulatory region to allow
transcription in the host, and any additional desired components, such as
reporter or marker genes, are cloned into the vector of choice using
standard cloning procedures in the art, such as described in Joseph
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs
Harbor 1989); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULAR BIOLOGY
(Wiley 1999), and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are
hereby incorporated by reference in their entirety. Once the nucleic acid
molecule encoding the peptide has been cloned into an expression vector,
it is incorporated into a host. Recombinant molecules can be introduced
into cells, without limitation, via transfection (if the host is a
eukaryote), transduction, conjugation, mobilization, or electroporation,
lipofection, protoplast fusion, mobilization, or particle bombardment,
using standard cloning procedures known in the art, as described by
JOSEPH SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold
Springs Harbor 1989), which is hereby incorporated by reference in its
entirety.

[0043] Recombinantly produced peptides of the invention can be purified by
several methods readily known in the art, including ion exchange
chromatography, hydrophobic interaction chromatography, affinity
chromatography, gel filtration, and reverse phase chromatography. The
peptide is preferably produced in purified form (preferably at least
about 80% or 85% pure, more preferably at least about 90% or 95% pure) by
conventional techniques. Depending on whether the recombinant host cell
is made to secrete the peptide into growth medium (see U.S. Pat. No.
6,596,509 to Bauer et al., which is hereby incorporated by reference in
its entirety), the peptide can be isolated and purified by centrifugation
(to separate cellular components from supernatant containing the secreted
peptide) followed by sequential ammonium sulfate precipitation of the
supernatant. The fraction containing the peptide is subjected to gel
filtration in an appropriately sized dextran or polyacrylamide column to
separate the peptides from other proteins. If necessary, the peptide
fraction may be further purified by HPLC.

[0044] Another aspect of the present invention relates to pharmaceutical
compositions containing the isolated peptides or fusion peptides of the
present invention. The pharmaceutical compositions may further comprise a
pharmaceutically acceptable excipient, carrier, buffer, stabilizer or
other material well known to those skilled in the art. Such materials
should be non-toxic and should not interfere with the efficacy of the
active ingredient. The precise nature of the carrier or other material
may depend on the route of administration, e.g., oral, intravenous,
cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal, patch
route, or other.

[0045] Pharmaceutical compositions for oral administration may be in
tablet, capsule, powder or liquid form. A tablet may include a solid
carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally include a liquid carrier such as water, petroleum,
animal or vegetable oils, mineral oil or synthetic oil. Physiological
saline solution, dextrose or other saccharide solution or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol may be included.

[0046] For intravenous, cutaneous or subcutaneous injection, or direct
injection at a tumor site, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and has
suitable pH, isotonicity and stability. Those of relevant skill in the
art are well able to prepare suitable solutions using, for example,
isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,
Lactated Ringer's Injection. Preservatives, stabilizers, buffers,
antioxidants and/or other additives may be included, as required.
Administration is preferably carried out to achieve delivery of a
therapeutically effective amount of the PRC2 inhibitory peptide. As used
herein, a "therapeutically effective amount" is the amount sufficient to
show benefit to the individual (i.e., a slowing or inhibition of cancer
progression). The actual amount administered, and rate and time-course of
administration, will depend on the nature and severity of cancer being
treated. Techniques for formulation and administration of the isolated
peptides of the present invention may be found in references well known
to one of ordinary skill in the art, such as Remington's "The Science and
Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins 2005.

[0047] The pharmaceutical compositions of the present invention can be
formulated for parenteral administration by bolus injection or continuous
infusion. Formulations for injection may be presented in unit dosage
form, e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or dispersing
agents. Alternatively, the active ingredient may be in powder form for
reconstitution before use with a suitable vehicle, e.g., sterile
pyrogen-free water.

[0048] In addition to the formulations described previously, the
pharmaceutical compositions of the present invention may also be
formulated as a depot preparation. Such long acting formulations may be
administered by implantation (for example, subcutaneously or
intramuscularly or by intramuscular injection). Thus, for example, the
pharmaceutical composition may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable oil)
or ion exchange resins, or as sparingly soluble derivatives (for example,
as a sparingly soluble salt). Additionally, the pharmaceutical
composition of the present invention may be delivered using a
sustained-release system, such as semi-permeable matrices of solid
hydrophobic polymers containing the composition. Various
sustained-release materials have been established and are well known by
those skilled in the art. Sustained-release capsules may, depending on
their chemical nature, release pharmaceutical composition for a few weeks
or up to over 100 days. Depending on the chemical nature and the
biological stability of the therapeutic composition, additional
strategies for protein stabilization may be employed.

[0049] Another aspect of the present invention relates to a method of
treating a subject having cancer that involves selecting a subject having
cancer and administering to the selected subject, an isolated peptide of
the present invention under conditions effective to treat the cancer.
Suitable isolated peptides of the present invention include peptides
comprising the amino acid sequence of SEQ ID NO: 4 (XAARMSXPXXG), SEQ ID
NO: 41 (TXAARMSXPXXGGVK), or SEQ ID NO: 44 (KQLATXAARMSXPXXGGVKK) as
described supra.

[0050] As used herein, a "subject" is any animal, preferably a mammal,
more preferably a human.

[0051] Since increased histone methylation via PRC2 complex activity has
been associated with certain cancers, a method for treating these types
of cancer in a subject involves administering to the subject a
therapeutically effective amount of an isolated peptide of the present
invention that inhibits methyltransferase activity, or restores
methyltransferase activity to roughly its level in counterpart normal
cells. In accordance with one embodiment of this aspect of the present
invention, the method of treating cancer in a subject involves
administering to the subject having cancer a therapeutically effective
amount of an isolated peptide of the present invention that inhibits
conversion of unmethylated H3-K27 to monomethylated H3-K27 (H3-K27me1).
In another embodiment of the present invention, the method of treating a
subject having cancer comprises administering to the subject in need
thereof a therapeutically effective amount of an isolated peptide of the
present invention that inhibits conversion of H3-K27me1 to dimethylated
H3-K27 (H3-K27me2). In another embodiment of the present invention, the
method of treating a subject having cancer involves administering to the
subject a therapeutically effective amount of an isolated peptide of the
present invention that inhibits conversion of H3-K27me2 to trimethylated
H3-K27 (H3-K27me3). In another embodiment of the present invention, the
method of treating a subject having cancer involves administering to the
subject a therapeutically effective amount of a compound that inhibits
both conversion of H3-K27me1 to H3-K27me2 and conversion of H3-K27me2 to
H3-K27me3. It is important to note that a disease-specific increase in
methylation can occur at chromatin in key genomic loci in the absence of
a global increase in cellular levels of histone or protein methylation.
For example, it is possible for aberrant hypermethylation at key
disease-relevant genes to occur against a backdrop of global histone or
protein hypomethylation.

[0052] Subjects particularly suitable for treatment in accordance with the
methods of the present invention have a cancer, leukemia, or lymphoma
that involves or is associated with aberrant PRC2 methyltransferase
activity. Cancers known to involve aberrant PRC2 methyltransferase
activity include, without limitation, leukemia (e.g., mixed-lineage
leukemia, acute myeloid leukemia, and chronic myelomonocytic leukemia),
lymphoma (e.g., follicular lymphoma and diffuse large B-cell lymphoma
(DLBCL)), breast cancer, melanoma, bladder cancer, gastric cancer,
endometrial cancer, prostate cancer, Ewing sarcoma, and non-small cell
lung cancer. Clinicians can readily identify whether a subject has a
cancer involving aberrant PRC2 methyltransferase activity by assaying
PRC2 activity or H3K27 methylation levels in a tumor or cancer cell
sample using methods well known in the art and described herein.
Alternatively, suitable subjects can be identified with genetic screening
for mutations in PRC2 proteins that are linked to increased PRC2 activity
and cancer, such as the Y641 mutation in EZH2 (Yap et al., "Somatic
Mutations at EZH2 Y641 Act Dominantly Through a Mechanism of Selectively
Altered PRC2 Catalytic Activity, to Increase H3K27 Trimethylation," Blood
117(8):2451 (2011), which is hereby incorporated by reference in its
entirety) or the A677 mutation in EZH2 (McCabe et al., "Mutation of A677
in Histone Methyltransferase EZH2 in Human B-Cell Lymphoma Promotes
Hypertrimethylation of Histone H3 on Lysine 27 (H3K27)," Proc. Nat'l.
Acad. Sci. USA 109(8):2989-94 (2012), which is hereby incorporated by
reference in its entirety).

[0053] In accordance with this aspect of the present invention, the
isolated PRC2 inhibitory peptides of the invention, or a pharmaceutical
composition containing the same, can be used in combination with another
anti-cancer therapeutic agent to treat cancer. The additional anti-cancer
therapeutic agent is typically an agent that is art-recognized as being
useful to treat the particular cancer being treated. The additional agent
also can be an agent that imparts a beneficial attribute to the
therapeutic peptide composition (e.g., an agent that affects the
viscosity of the composition).

[0054] The isolated peptides, including fusion peptides, of the present
invention or pharmaceutical composition containing the same can be
administered simultaneously or sequentially with the additional
therapeutic agent. In one embodiment, the combination therapy is
formulated into a single pharmaceutical composition to achieve
simultaneous administration. Alternatively, separate compositions
comprising the PRC2 inhibitory peptides and the one or more additional
cancer therapy agent(s) can be co-administration.

[0056] As used herein, a "therapeutically effective amount" or
"therapeutically effective dose" is the dose of one or more isolated PRC2
inhibitory peptide(s) that inhibits, totally or partially, the
progression of the cancerous condition or alleviates, at least partially,
one or more symptoms of the cancerous condition. The dosage of peptide
that is therapeutically effective will depend upon the patient's size and
gender, the cancer to be treated, the severity (i.e., stage) of cancer
condition, and the result sought. In one embodiment, a therapeutically
effective dose refers to that dosage of a PRC2 inhibitory peptide that
results in amelioration of cancer symptoms in a patient. For a given
patient, a therapeutically effective amount may be determined by methods
known to those of skill in the art.

[0057] Toxicity and therapeutic efficacy of PRC2 inhibitory peptides can
be determined by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., for determining the maximum tolerated dose
(MTD) and the ED50 (effective dose for 50% maximal response). The
dose ratio between toxic and therapeutic effects is the therapeutic index
and it can be expressed as the ratio between MTD and ED50. The data
obtained from cell culture assays and animal studies can be used in
formulating a range of dosage for use in humans. Dosage may also be
guided by monitoring the inhibitory peptide's effect on pharmacodynamic
markers of enzyme inhibition (e.g., histone methylation or target gene
expression) in diseased or surrogate tissue. Cell culture or animal
experiments can be used to determine the relationship between doses
required for changes in pharmacodynamic markers, and doses required for
therapeutic efficacy can be determined in cell culture or animal
experiments or early stage clinical trials. A suitable dosage of a PRC2
inhibitory peptide lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity. The
dosage may vary within this range depending upon the dosage form employed
and the route of administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician in
view of the patient's condition.

[0058] Dosage amount and interval may be adjusted individually to provide
plasma levels of the active moiety which are sufficient to maintain the
methyltransferase modulating effects, or minimal effective concentration
(MEC) for the required period of time to achieve therapeutic efficacy.
The MEC will vary for each inhibitory peptide but can be estimated from
in vitro data and animal experiments. Dosages necessary to achieve the
MEC will depend on individual characteristics and route of
administration. However, high pressure liquid chromatography (HPLC)
assays or bioassays can be used to determine plasma concentrations.

[0059] Dosage intervals can also be determined using the MEC value. In
certain embodiments, PRC2 inhibitory peptides should be administered
using a regimen which maintains plasma levels above the MEC for 10-90% of
the time, preferably between 30-90% and most preferably between 50-90%
until the desired therapeutic effect is achieved. In other embodiments,
different MEC plasma levels will be maintained for differing amounts of
time. In cases of local administration or selective uptake, the effective
local concentration of the drug may not be related to plasma
concentration.

[0060] One of skill in the art can select from a variety of administration
regimens and the amount inhibitory peptide administered will, of course,
be dependent on the subject being treated, on the subject's weight, the
severity of the affliction, the manner of administration and the judgment
of the prescribing physician.

[0061] Another aspect of the present invention relates to antibodies that
immunospecifically-bind H3.3 and/or H3.1 proteins and peptides thereof
containing the K27M mutation. Preferably these antibodies are generated
using the isolated peptides of the present invention as immunogens.

[0062] As used herein, the term "antibody" is meant to include intact
immunoglobulins derived from natural sources or from recombinant sources,
as well as immunoreactive portions (i.e., antigen binding portions) of
intact immunoglobulins. The antibodies of the present invention may exist
in a variety of forms including, for example, polyclonal antibodies,
monoclonal antibodies, intracellular antibodies ("intrabodies"), antibody
fragments (e.g. Fv, Fab and F(ab)2), as well as single chain antibodies
(scFv), chimeric antibodies and humanized antibodies (Ed Harlow and David
Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor
Laboratory Press, 1999); Houston et al., "Protein Engineering of Antibody
Binding Sites: Recovery of Specific Activity in an Anti-Digoxin
Single-Chain Fv Analogue Produced in Escherichia coli," Proc Natl Acad
Sci USA 85:5879-5883 (1988); Bird et al, "Single-Chain Antigen-Binding
Proteins," Science 242:423-426 (1988), which are hereby incorporated by
reference in their entirety).

[0063] Methods for monoclonal antibody production may be carried out using
techniques well-known in the art (MONOCLONAL ANTIBODIES--PRODUCTION,
ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M.
Ladyman eds., 1995), which is hereby incorporated by reference in its
entirety). Generally, the process involves obtaining immune cells
(lymphocytes) from the spleen of a mammal which has been previously
immunized with an isolated peptide of the present invention either in
vivo or in vitro. Exemplary isolated peptides are described supra. In one
embodiment of this aspect of the present invention, the antibody is
raised against an isolated peptide comprising the amino acid sequence of
AARMSAPSC (SEQ ID NO: 55) or AARMSAPAC (SEQ ID NO: 56).

[0065] The antibody-secreting lymphocytes are then fused with myeloma
cells or transformed cells, which are capable of replicating indefinitely
in cell culture, thereby producing an immortal, immunoglobulin-secreting
cell line. Fusion with mammalian myeloma cells or other fusion partners
capable of replicating indefinitely in cell culture is achieved by
standard and well-known techniques, for example, by using polyethylene
glycol (PEG) or other fusing agents (Milstein and Kohler, "Derivation of
Specific Antibody-Producing Tissue Culture and Tumor Lines by Cell
Fusion," Eur J Immunol 6:511 (1976), which is hereby incorporated by
reference in its entirety). The immortal cell line, which is preferably
murine, but may also be derived from cells of other mammalian species, is
selected to be deficient in enzymes necessary for the utilization of
certain nutrients, to be capable of rapid growth, and have good fusion
capability. The resulting fused cells, or hybridomas, are cultured, and
the resulting colonies screened for the production of the desired
monoclonal antibodies. Colonies producing such antibodies are cloned, and
grown either in vivo or in vitro to produce large quantities of antibody.

[0066] Procedures for raising polyclonal antibodies are also well known.
Typically, such antibodies can be raised by administering the isolated
peptides of the present invention subcutaneously to rabbits (e.g., New
Zealand white rabbits), goats, sheep, swine, or donkeys which have been
bled to obtain pre-immune serum. The antigens can be injected in
combination with an adjuvant. The rabbits are bled approximately every
two weeks after the first injection and periodically boosted with the
same antigen three times every six weeks. Polyclonal antibodies are
recovered from the serum by affinity chromatography using the
corresponding antigen to capture the antibody. This and other procedures
for raising polyclonal antibodies are disclosed in Ed Harlow and David
Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor
Laboratory Press, 1988), which is hereby incorporated by reference in its
entirety.

[0067] Methodologies for the screening of antibodies that possess the
desired specificity include, but are not limited to, enzyme-linked
immunosorbent assay (ELISA), western blot, and other
immunologically-mediated techniques known within the art.

[0068] The antibodies of the present invention may be used in cancer
diagnostic and prognostic methods. The lysine to methionine mutation at
amino acid residue 27 of H3 has been implicated in high grade pediatric
gliomas, including supratentorial glioblastomas (GBM) and diffuse
intrinsic pointine gliomas (DIPG) (see e.g., Schwartzentruber et al.,
"Driver Mutations in Histone H3.3 and Chromatin Remodelling Genes in
Paediatric Glioblastoma," Nature 482(7384):226-31 (2012), which is hereby
incorporated by reference in its entirety). The presence of this mutation
has also been linked to reduced survival in DIPG (Khuong-Quang et al.,
"K27M Mutation in Histone H3.3 Defines Clinically and Biologically
Distinct Subgroups of Pediatric Diffuse Intrinsic Pontine Gliomas," Acta
Neuroathol. 124(3):439-47 (20120), which is hereby incorporated by
reference in its entirety), and its detection can be used to determine a
patient's treatment regimen (e.g., patients with the K27M mutation may be
started on a more aggressive treatment regimen early during the course of
the disease).

[0069] Accordingly, another aspect of the present invention relates to a
method of diagnosing or prognosing a subject having cancer. This method
involves detecting, in the subject having cancer, the presence of the
K27M mutation in H3 using a diagnostic reagent, where the diagnostic
reagent is an K27M H3 antibody, or active binding fragment thereof, of
the present invention. As described supra, the antibody has antigenic
specificity for the K27M mutation of H3.3 and H3.1. The diagnosis or
prognosis of the subject is based on the detection of H3 K27M mutation in
the subject.

[0070] Detecting the presence of the H3 K27M mutation in the subject using
the diagnostic antibody reagent of the present invention can be achieved
by obtaining a biological sample from the subject (e.g., tumor,
bone-marrow, blood), contacting the biological sample with the diagnostic
antibody reagent, and detecting binding of the diagnostic antibody
reagent to H3 K27M mutant protein in the sample from the subject. Assays
suitable for detecting the H3 K27M mutation in patient samples include,
without limitation, western blot, immunohistochemistry, and ELISA, all of
which are well known to those of skill in the art.

[0077] The H3K27M mutation identified in a majority of pediatric DIPGs
occurs at a well-studied residue on the H3 N-terminal tail. H3K27 is
subject to both acetylation (K27ac) by CBP/P300, and to varying degrees
of methylation (K27me1/2/3) by PRC2. The K27M mutation will block
post-translational modification on these mutant histone H3 proteins,
however, it is unknown how this mutant histone will impact chromatin
structure. The heterozygous appearance of H3F3A or HIST3H1B missense
mutation at K27, and the exclusive lysine to methionine substitution
suggests a gain-of-function for H3 proteins that contain K27M.

[0078] Antisera that specifically recognizes the K27M substitution in both
H3.1/2 and H3.3 contexts was generated (FIGS. 1A-1D). Using this
antibody, acid extracted histones from human DIPG tumors, some of which
contained the K27M genetic lesions in H3F3A (H3.3) or HIST3H1B (H3.1)
were probed Immunoblots with the K27M-specific antibody on these DIPG
histone samples indicated the presence of H3K27M protein (FIG. 1A).
Whether DIPG samples that contain the K27M mutation have global changes
in histone modification status was also determined. Immunoblots with
modification-specific antisera showed that DIPG tumors that contain K27M
mutations exhibited decreased H3K27me3, and also a modest increase in
levels of H3K27ac (FIG. 1A). The quantities of two
transcription-activation related histone modifications (H3K4me3 and
H3K36me3) were similar in DIPG samples regardless of tumor genotype.

[0079] Whether the presence of H3K27M lead to the striking reduction in
H3K27me3 signal in the DIPG samples was then determined. To this end,
stable HEK293T cell lines that express FLAG and HA epitope-tagged histone
H3.3 or pediatric glioma H3.3 mutants K27M were generated. Additionally,
HEK293T cells that express H3.3G34R or G34V, two H3.3-specific missense
mutations identified in pediatric GBMs were also generated. Immunoblot on
whole cell extracts showed that cells expressing the K27M mutant histone
exhibited a decrease in overall K27me3 and K27me2 levels, but showed no
change in H3K4me3 or H3K36me3 levels (FIG. 1B). Interestingly, the global
loss of H3K27me2/3 was specific to the H3K27M mutation, as no reduction
was observed with H3K27R or H3K27Q.

[0080] The global reduction in H3K27me2/3 signal suggested that the H3K27M
exogenous transgene reduced methylation on endogenous wildtype H3.
Chromatin containing H3K27M was purified to examine the
post-translational modification status of endogenous histone H3.
Mononucleosome populations (>95%) that contain the epitope-tagged H3.3
or K27M were purified by immunoprecipitation with M2-FLAG resin after
extensive digestion with micrococcal nuclease. Ponceau stain of
immunoprecipitated mononucleosomes resolved by SDS-PAGE show a near 1:1
ratio of epitope-tagged H3.3 to the faster migrating endogenous H3 (FIG.
1C). The post-translational modification status of histone H3.3 in the
immunoprecipitated mononucleosomes was probed. Previous work showed that
exogenously expressed epitope tagged H3.3 associates exclusively with
endogenous H3.3 (Loyola et al., "PTMs on H3 Variants Before Chromatin
Assembly Potentiate their Final Epigenetic State," Mol Cell 24:309-316
(2006), which is hereby incorporated by reference in its entirety) and it
was found that the modification status of the endogenous H3.3 versus the
tagged-H3.3 in wildtype nucleosomes was nearly equal for H3K27me3,
H3K27ac and H3K36me3 (FIG. 1C). Mononucleosomes that contained an average
of one epitope-tagged H3.3K27M histone exhibited a decrease in H3K27me3
on the endogenous H3.3 protein. While these nucleosomes exhibited a
decrease in H3K27me3, they showed an increase in the acetylation of H3K27
(H3K27ac) (FIG. 1C). The H3K36me3 levels on the epitope-tagged G34R and
G34V histones decreased, however, the levels on the endogenous histone
H3.3 remained unchanged (FIG. 1C).

[0081] Oligonucleosomes from HEK293T cell lines were purified by limited
microcoocal nuclease digestion (>95% of 4-5 nucleosomes in length,
with a mode of 2-3 nucleosomes). Ponceau stain of core histones from
these arrays suggested the ratio of epitope-tagged H3.3 to endogenous H3
was less than one, indicating that some nucleosomes within the
immunoprecipitated arrays contained only endogenous H3 (FIG. 1D). Similar
to the mononucleosomes, an increase in the amount of H3K27ac was observed
in arrays containing H3.3K27M.

[0082] While about 43% of pediatric brain tumors contain the K27M mutation
in the H3F3A gene, almost 12% of cases contained the K27M mutation in one
gene (HIST1H3B) of the 12 different genes that encode H3.1 (Wu et al.,
"Somatic Histone H3 Alterations in Pediatric Diffuse Intrinsic Pontine
Gliomas and Non-brainstem Glioblastomas," Nat Genet 44: 251-253 (2012),
which is hereby incorporated by reference in its entirety).
Oligonucleosomes purified from HEK293T cells that express epitope-tagged
H3.1 also exhibited a decrease in the H3K27me3 signal on neighboring
nucleosomes (FIG. 1D).

[0083] The invariant nature of the lysine 27 to methionine mutation in
nearly 80% of pediatric DIPGs suggests that this amino acid substitution
imparts a unique gain-of-function to the mutant histone. A series of
amino acid substitutions at H3K27 were constructed to determine if the
inhibition of K27me3 on neighboring nucleosomes was unique to K27M.
Oligonucleosomes containing K27R (an unmethylatable basic residue) or
K27Q (a acetyl-lysine mimic) showed no difference in H3K27me3 on the
endogenous H3.3 in immunoprecipitated oligonucleosomes arrays (FIG. 1D).
The K27A mutation showed a modest decrease in K27me3 levels on the
endogenous H3.3, though reproducibly higher than the K27M mutation.

[0084] The immunoprecipitated mono- and oligonucleosomes from HEK293T
cells expressing epitope tagged-G34R or G34V exhibited little difference
in K27 acetylation or trimethylation. However, these nucleosomes did
exhibit a decrease in H3K36m3 on only the epitope-tagged H3.1/3 in either
mono- or oligonucleosomes as measured by immunoblot (FIG. 1C, 1D). The
observed decrease in H3K36me3 signal on tagged H3G34R/V may result from
the destruction of K36me3 antibody epitope. Indeed, the G34R or G34V
mutants decreased the immunoblot signal of the H3K36me3 antibody by
nearly 10 fold on H3 peptides. Mass spectrometry was used to examine the
modification status on H3K36 from immunoprecipitated mononucleosomes. The
epitope-tagged H3 exhibited a decrease in K36me2 (˜2.5 fold) and
K36me3 (˜20 fold) on the G34R/V mutants compared to wildtype.

Example 2

H3K27M-Containing Chromatin is a Poor Substrate for PRC2

[0085] The decrease in H3K27me2/3 in chromatin containing H3K27M suggests
that this template may be a poor substrate for PRC2. To test this
hypothesis, recombinant PRC2 was purified from Hela or HEK293T cells that
express a FLAG-tagged EED transgene (FIG. 2A). Using purified PRC2 and
mono- or oligonucleosomes purified from HEK293T cells (FIG. 2B), histone
methyltransferase reactions were performed using radiolabeled SAM.
Previous studies found that K27me3 peptides could stimulate PRC2
methyltransferase activity on nucleosome substrates (Margueron et al.,
"Role of the Polycomb Protein EED in the Propagation of Repressive
Histone Marks" Nature 461: 762-767 (2009), which is hereby incorporated
by reference in its entirety). Allosteric activation of PRC2 activity by
the product of its own catalytic activity provides an attractive
mechanism for the replication of this important histone modification
linked to epigenetic gene silencing. Incubation of 10 or 100 μM of
K27me3 peptide strongly stimulated PRC2 activity towards mononucleosome
or oligonucleosome templates (FIG. 2C). PRC2 activity on mononucleosome
templates that contained on average one epitope tagged H3.3K27M was
assessed. Very little PRC2-dependent methylation was detected on the
endogenous wildtype H3.3 protein found in K27M mononucleosomes (FIG. 2C).
Similarly, oligonucleosomes containing K27M also showed decrease
PRC2-dependent methylation as compared to wildtype H3.3 oligonucleosomes.

[0086] The K27M mutation uniquely decreased the levels of H3K27me3 on
nucleosome arrays immunoprecipitated from 293T cells (FIG. 1C, D).
Methyltransferase assays on different mononuclesome templates were
performed to determine if the reduced PRC2 activity on H3.3 present in
FIG. 2C was unique to K27M mononucleosomes. Mononucleosomes containing
the K27M mutation exhibited the least amount of methylation as compared
to templates with K27Q, K27R or K27A (FIG. 2D). However, mononucleosomes
with K27R, K27Q or K27A showed a modest reduction in methylation on the
endogenous H3.3 protein relative to wildtype H3.3 mononucleosomes.

Example 3

H3K27M Peptide Inhibits PRC2 Activity in Trans

[0087] The reduced methylation on nucleosome templates suggests that the
K27M peptide may interfere with PRC2 activity. Previously, peptides from
histone H1K26me3 were shown to inhibit PRC2 methyltransferase activity on
nucleosome templates in trans (Xu et al. "Binding of Different Histone
Marks Differentially Regulates the Activity and Specificity of Polycomb
Repressive Complex 2 (PRC2)," Proc Natl Acad Sci USA 107: 19266-19271
(2010), which is hereby incorporated by reference in its entirety).
Whether H3.3K27M peptides could interfere with PRC2 activity when added
in trans was determined. The K27me3 peptide strongly stimulated PRC2
activity, whereas unmodified or K27ac peptide exhibited little
stimulation relative to the no peptide control (FIG. 3A). Incubation of
K27M peptide decreased PRC2 activity on nucleosomes to below the no
peptide signal (FIG. 3A). Incubation of this same set of peptides using a
H3.3K27M-containing nucleosome template showed little or undetectable
methylation on the endogenous H3.3 (FIG. 3B).

[0088] An increasing concentration of K27M or K27ac peptide was titrated,
while the concentration of K27me3 peptide was simultaneously decreased. A
modest decrease in PRC2 activity at a K27M:K27me3 ratio of 1:2.3 was
observed that steadily grew with an increased ratio of K27M to K27me3
peptide. In contrast, no decrease in PRC2 activity was observed at a 9:1
ratio of K27ac: K27me3 peptide (FIG. 3D, 3E). The inhibitory effect of
the K27M peptide became more pronounced with titration of K27M into PRC2
reactions containing a constant low concentration of K27me3 peptide (FIG.
3F). Again, no decrease in PRC2 activity was observed with a titration of
the K27ac peptide. These data confirm that the K27M peptide is an
inhibitor of PRC2 activity.

[0089] Kinetic studies were performed to better understand the inhibitory
effect of the K27M peptide on PRC2 activity. Time course of PRC2
methyltransferase assays with varying mononucleosome substrate
concentrations were performed without or with K27M peptide at two
different concentrations (FIG. 4A). Plotting the initial velocity versus
nucleosome substrate concentration showed a reduction in the apparent
Vmax by addition of the K27M peptide. PRC2 reactions that contained
H3K27ac peptide of identical peptide concentrations did not decrease the
apparent Vmax of the reaction. A double reciprocal Lineweaver-Burke plot
of the initial velocity versus substrate plot showed that H3K27M
primarily affects the Vmax of the reaction, while having little effect on
the Km of the substrate. These data are consistent with a non-competitive
inhibition model for the H3K27M peptide. The Ki for the H3K27M was
calculated to be 21 μM.

Example 5

Inhibition of H3K27 Methylation is Specific to the H3K27M Containing
Peptide

[0090] The heterozygous and invariant nature of the lysine-to-methionine
mutation at residue 27 in nearly 80% of pediatric DIPGs strongly suggests
that this specific amino acid substitution imparts a unique
gain-of-function to the mutant histone. To further test the specificity
of this substitution, a survey of all amino acid substitutions at H3K27
was performed. Nearly all substitutions had little effect, if any, on the
amounts of K27me3, with the exception of methionine, and to a lesser
extent isoleucine (FIG. 5A). Titration of K27M peptide to in vitro
methylation reactions revealed a median inhibitory concentration
(IC50) of 5.9 μM [95% confidence interval (CI) of 1.10 to 6.42].
Peptides containing Lys27 replaced by Ile inhibited PRC2 to a lesser
extent than K27M (IC50 for K27I=8.9 μM (95% CI: 4.12 to 11.2),
whereas Lys27 replaced by Leu had no inhibitory effect on the
amounts of H3K27me3 in vivo or PRC2 in vitro (FIGS. 5B and 5C). To
evaluate whether the thioether moiety of methionine was required for
inhibition of PRC2, a norleucine derivative (K27N1e) was prepared. The
K27Nle variant proved to be an even better inhibitor of PRC2. (IC50
for K27Nle=0.85 μM) (95% CI: 0.57 to 1.27) (FIG. 5D). Thus, a long,
hydrophobic residue suffices for EZH2 binding, and methionine--and to a
slightly lesser extent isoleucine--represents the ideal biochemically
accessible choices.

[0091] To carry out the experiments described above, human 293T, 293 or
murine PDGF-transduced glioblastoma cells were transduced with
recombinant, concentrated Lentivirus made with the pCDH-EF1-MCS-Puro or
Neo expression vector (5 μg/mL polybrene, 2×10 7 IFU).
Transduced cells were grown under selection (1 μg/mL Puromycin or 0.8
μg/mL G418) at 24 hours post transduction for 72 hours. Cells were
collected at 7-10 days post transduction for immunoblot analysis. The
amino acid sequence of epitope tagged-H3.3 used in transgenic experiments
is shown below as SEQ ID NO: 57. The C-terminal FLAG and HA epitope
sequences are shown in bold.

[0092] Although preferred embodiments have been depicted and described in
detail herein, it will be apparent to those skilled in the relevant art
that various modifications, additions, substitutions, and the like can be
made without departing from the spirit of the invention and these are
therefore considered to be within the scope of the invention as defined
in the claims which follow.